JP6932861B2 - Proteome assay using quantum sensor - Google Patents

Description

The present invention relates to the simultaneous detection of hundreds or thousands of proteins in biological fluids such as blood or urine in the first step of proteome analysis. In particular, the present invention uses a label-free assay that achieves the detection without the use of mass spectrometry or chromatographic methods and allows the use of compact, inexpensive and reusable instruments. ..

Traditionally, various attempts to assess gene activity or to decipher biological processes, including disease processes or biological processes of pharmacological action, have focused on genes. However, proteomics can provide additional information about the biological functions of cells and organisms. Proteomics involves qualitative and quantitative measurements of gene activity by detecting and quantifying expression at the protein level rather than at the gene level. Proteomics also includes the study of genetically unencoded events such as post-translational modifications of proteins and interactions between proteins.

Currently, a large amount of genomic information is available. DNA chips have been practically used as molecular arrays for this purpose, and the price of direct DNA sequences continues to decline significantly. In addition, there is an increasing demand for high-throughput proteomics. Chips have been proposed to detect proteins that are more complex and variable in biological function than DNA, which is currently the subject of intensive research for many applications. Proteomics is much more preferred than the genome for health monitoring, where the genome is static and represents only medical potential, whereas proteomes dynamically fluctuate depending on the patient's condition and even This is because it is said to define those pathological conditions. However, it is difficult to detect and quantify proteins, whereas it is relatively easy to detect and quantify nucleic acids. This motivated many attempts to measure the concentration of mRNA (messenger RNA) instead of protein concentration. Unfortunately, it has been shown that mRNA levels do not correlate well with protein levels. Proteomics will inevitably depend on the ability to detect proteins directly.

A "protein chip" is a set used to refer to any device in which a protein or molecule (capture reagent) for capturing the protein is immobilized on the surface of the chip, allowing detection of protein binding. Terminology. Until recently, capture reagents on protein chips were predominantly antibodies. Detection of specific proteins in complex mixtures such as biofluids requires high specificity, and many protein chips that utilize antibodies also rely on sandwich assays to boost specificity. Such assays are known to have important drawbacks for proteomics, some of which are protein and / or antibody specific, and some of which are sandwich assay specific. ..

Currently, there is no economically viable means of collecting proteomics data from human subjects on a regular basis. Measurement of sample proteomics is usually achieved by various chromatographic techniques for sample preparation combined with various mass spectrometric techniques for detection and quantification. The instruments used in these procedures are expensive and large, so samples usually have to be shipped to a central processing facility. The need to transport a sample requires that the sample be processed at the time of collection for storage during shipping or stored in place until it can be shipped. Unfortunately, preparation and storage protocols tend to vary widely from place to place, and even within the same place. Differences in these protocols always result in significant variations downstream of proteomics measurements, making analysis difficult or impossible.

The ideal proteomics collector approaches working in a completely solid state (with few moving parts) and minimizes costs by utilizing label-free detection technology to minimize reagent use. It is reusable for limited, endless measurements and operates with small amounts of biofluid. Fluctuations in sample analysis due to variations in sample preparation and storage protocols eliminate sample storage and transport by minimizing or eliminating sample preparation and taking sample measurements in place, time and collection. By doing so, it can be reduced.

A new class of non-protein-based capture reagents has been found in nucleic acid molecules. A long-standing theory has been that nucleic acids have an informational role in the first place. Through a method known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment), sometimes also referred to as the SELEX process, it has become clear that nucleic acids have three-dimensional structural diversity that is not very different from proteins. There is. The SELEX process is a method for in vitro evolution of nucleic acid molecules that bind very specifically to a target molecule, each of which is incorporated by reference in the present disclosure, filed June 11, 1990. U.S. Patent Application No. 07/536,428 of the title "Systematic Evolution of Ligands by EXPONENTIAL Enrichment," now abandoned U.S. Patent No. 5,475,096 (Title "Nucleic Acid Litens"), U.S.A. It is described in Nos. 5,270,163 (also see International Patent Publication Nos. 91/19813) (title "Nucleic Acid Lights"). Each of these publications, collectively referred to herein as a SELEX patent application, describes a method for making a nucleic acid capture reagent for any desired target molecule.

The SELEX process provides a class of products called nucleic acid ligands or aptamers, each of which has a unique sequence and the property of specifically binding to a desired target compound or molecule. Each nucleic acid capture reagent identified by SELEX is a specific ligand for a given target compound or molecule. The SELEX process is available within a nucleic acid to act as a ligand (form a specific binding pair) with virtually any chemical compound, whether it is a monomer or a polymer. It is based on the unique finding that it has sufficient properties and sufficient chemical versatility to form various two-dimensional and three-dimensional structures. Molecules of any size or composition can act as targets.

Suitable for the application of high affinity binding, the SELEX method uses the same general selection scheme to achieve virtually any desired criterion of binding affinity and selectivity, from a mixture of candidate oligonucleotides. Includes the selection of, and the stepwise repetition of binding, separation, and amplification. Starting with a mixture of nucleic acids, preferably nucleic acids containing segments of randomized sequences, the SELEX method involves contacting the target with the mixture under favorable conditions for binding and specifically binding to the target molecule. After separating the unbound nucleic acid from the existing nucleic acid, dissociating the nucleic acid-target complex, and amplifying the dissociated nucleic acid from the nucleic acid-target complex to produce a ligand-rich nucleic acid mixture. It includes a step of binding, a step of separating, a step of dissociating, and a step of amplifying through a cycle as needed to generate a nucleic acid ligand having a highly specific affinity for the target molecule.

SOMAmer (Slow Off-late Modified Aptamers) is an aptamer with improved off-rate characteristics. This improvement in dissociation characteristics _{can be expressed as the dissociation rate (t 1/2} ) or the time at which 50% of the aptamer / target complex is dissociated. Such dissociation rates are generally 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, and 240 minutes. It can vary from super-variable, which is the average time for the protein-aptamer complex to dissociate. In addition, SOMAmer contains modified nucleosides that provide different built-in functionality. These functionalities may include tags for immobilization, labels for detection, means for promoting or controlling separation, hydrophobic side chains for providing good affinity with proteins, and the like. The modification to improve the affinity with the protein is the chemical group attached to the 5-position of the pyrimidine base. The addition of a functional group at the 5-position (eg, with a benzyl group, naphthyl group, or indole group) expands the chemical diversity of SOMAmer, allowing high affinity binding to a wider range of target molecules. .. In addition, some polymerases can further transcribe DNA with modifications of these positions, thus allowing the amplification required for the SELEX process. SOMAmer and the methods for making these are incorporated herein by reference, respectively, in US Pat. Nos. 7,964,356 and 7,947,447 (both titles are "Metaphod for". It is described in "generating aptamers with improbed off-rates").

It should be noted that although aptamers and SOMAmers can be found by the SELEX process, there may be other means for selecting them. For example, due to the improved computer modeling of molecular interactions, the ideal nucleic acid sequence for aptamers and the relevant chemical modifications for SOMAmer can be directly calculated to create capture reagents specific for a given target molecule. Can be possible. Other chemical techniques for screening aptamers and SOMAmers other than SELEX are also possible.

Assays aimed at detecting and quantifying physiologically important molecules in biological and other samples are important tools in the field of scientific research and healthcare. Other classes of such assays include the use of microarrays containing one or more aptamers immobilized on a solid carrier. Each of these aptamers can bind to the target molecule in a highly specific manner and with a very high affinity. For example, U.S. Pat. Nos. 5,475,096 (titled "Nucleic Acid Ligands"), as well as 6,242,246, 6,458,543, and 6,503,71 (6,503,71). Please refer to (the title of each is "Nucleic Acid Ligand Digital Biochip"). These patents are incorporated herein by reference in this disclosure. When the microarray is contacted with the sample, the aptamer binds to each target molecule present in the sample, which can determine the absence, presence, amount, and / or concentration of the target molecule in the sample.

Label-free assays are considered highly desirable, but not always achievable. A label is any exogenous molecule that is chemically or temporarily attached to a molecule of interest to detect the presence or activity of the molecule. Label-free assays utilize the biophysical properties of a molecule, such as molecular weight, charge of the molecule, relative permittivity, or (in the case of the present invention) affinity for aptamers, to monitor the presence or activity of the molecule. do. Some embodiments of the invention utilize spin labels associated with surface-attached aptamers. The present invention is considered a label-free assay because the spin label is attached to a component of the detection system (eg, aptamer) as opposed to the molecule of interest (eg, protein). Many sensitive assays require that the analyte be "labeled" with a detectable tag. This tag or label can be a dye, a radioisotope, or any other readily measurable, which can be used to measure the analyte on its behalf.

ELISA assays (enzyme-linked immunosorbent assay) are considered "absolute criteria" for immunoassays due to their high sensitivity and specificity, but are not considered label-free. ELISA is a "sandwich assay" as opposed to label-free, using two antibodies specific for different binding points (epitope) on the analyte. One of the antibodies is immobilized on the surface and captures the analyte from the sample solution. The second antibody is linked to an enzyme that catalyzes a detectable change in a particular additive. After the analyte has been captured by the immobilized antibody, the surface is washed to remove unwanted molecules, a second antibody is added, then the wash is performed, and the addition is usually catalyzed to a detectable dye. Add the agent. The second antibody imparts specificity and detectability, but it also requires several steps involving costly reagents. The label-free form of this assay consists of only a first antibody and some means of detecting the binding of the analyte.

Biochips, including protein chips, require a means of detecting specific analytes in a sample, ignoring other molecules. The detectors and methods described by this technique are based on color centers located near the surface of a solid that can be probed through Optically Detect Magnetic Resonance (ODMR) or other techniques. The color center is a point defect close to an ideal transparent crystalline insulator such as diamond, silicon carbide, or silica or a semiconductor with a large bandgap. These can consist of substitution defects in which atoms in the crystal are replaced by other types of atoms, vacancy defects in which no atoms are present, or a combination of the two.

The color center has a localized electron orbital that resembles a free atom. This electronic state is ordered in terms of principal quantum number, orbital quantum number, and magnetic quantum number, and can be stable when charged, neutral, or both. A wide bandgap or insulating crystal surrounding the color center acts as a "vacuum" that separates the color center. At a sufficiently low density, this is abundant and well-suited with discrete optical transitions in the visible region that coexist with electron / nucleus spin states with long relaxation times (hence the name "color center"). It results in an independent "atomic-like" entity with a split composite energy spectrum.

Of particular interest is the color center whose fluorescence intensity depends on the spin polarization state (ie, the ground state magnetic quantum number). In this case, the lower level set of magnetism is reflected in the fluorescence intensity, allowing optical detection of magnetic resonance. Because ODMR essentially translates radiofrequency (RF) or microwave frequency detections from lower-level transitions of optics into much higher energy photon detection, it is in sensitivity (about 5 orders of magnitude) and It has obvious advantages with respect to strong fluorescence, allowing optical observation of individual color centers.

An example of a color center where such ODMR is possible is nitrogen-vacancy (NV) in diamond crystals. The preferred embodiment described in the present invention utilizes NV centers in diamond for detection, but the present invention can also use other color centers. Diamond itself has more than 500 known color centers and is mostly associated with nitrogen. Other elements known as possible substitutions in the diamond lattice include nickel, boron, silicon, hydrogen, and cobalt. Color centers in other crystal lattices include, for example, germanium-related defects in germanium silicate glass, silicon defects in silicon carbide, and X-ray-induced _{defects in LiBaF 3 crystals.}

As the name implies, the NV center consists of nitrogen substitutions for carbon atoms located next to adjacent vacancies in the lattice. This is shown in FIG. 1, which is a schematic representation of a diamond crystal, generally comprising an NV center represented by 100 and 104. The NV center is a paramagnetic color center with a unique coupling between the spin state and the optical state of the electron. It can emit strong and stable fluorescence (ie a large absorption coefficient combined with a short lifetime excited state), also exhibits a very long magnetic relaxation time, and is sensitive to local properties such as magnetic or electric fields. Become a detector. The diamond itself is very stable mechanically, thermally and chemically. At the same time, the surface of diamond is suitable for chemical modification, which is useful for binding molecular agents. Pure diamond is optically transparent, allowing free excitation and emission of the fluorescence center, which fluorescence also exhibits no photobleaching and minimal luminescence intermittentness (“blinking”). The combination of all these properties makes NV centers a good candidate for highly sensitive biosensing.

The present disclosure is for detecting a target molecule in a sample based on changes in the properties of one or more color sensors located near the surface of the substrate as the target molecule binds to a surface-adhered capture reagent. Describe the device, system, and method. The detector can be configured to detect the target molecule in the sample by detecting changes in the nature of the color center. In some cases, the target molecule is a protein and the capture reagent is an aptamer. In some cases, the color center is the NV center located in the diamond, and the change in properties is the change in fluorescence radiation. In some cases, a large number of protein species, such as hundreds, thousands, tens of thousands, or even more, can be targeted in one assay.

The technique involves the attachment of aptamers or SOMAmers on surfaces such as diamond surfaces, and the passivation of areas of the surface that do not bind aptamers or SOMAmers to non-specific binding by unwanted proteins. The technique is also involved in the regeneration of surface-attached aptamers or SOMAmers for a few more rounds of protein detection.

The technique aims to eliminate all moving moieties except those associated with the delivery of large amounts of fluid to the active surface of the biochip. These fluids include sample fluids, cleaning fluids, and fluids used for biochip regeneration. The proposed detection scheme is a label-free method, meaning that no detection agent needs to be added to the sample to allow measurement of the protein of interest. Regeneration of the active surface is non-destructive and requires only a mild buffer wash to dissociate the protein from the aptamer or SOMAmer. The NV center is extremely stable, and the aptamer and SOMAmer itself are very stable and can be used hundreds of times.

More specifically, some time is required for the protein to bind to the immobilized aptamer after the sample solution has come into contact with the active surface. This binding time also includes the time required for the protein to diffuse from the bulk fluid to the surface, which can take several hours. During this binding time, the sample fluid can be static, but it is more common to agitate or recirculate the fluid to bring unbound proteins closer to the surface, thereby reducing the distance that needs to be diffused. be. After the binding step is complete, it is common to wash the surface to remove unbound proteins. This washing is generally performed with a buffer solution similar to the sample solution, such as phosphate buffer, or distilled water. If highly specific detection is required, washing may be done by using a mild denaturing agent such as high salt concentration, low concentration (<1M) urea, or by including a competitor to the protein of interest. Can be strengthened. Such competitors may be, for example, albumin, which competes with proteins, or heparin, which competes with nucleic acids. Nucleic acids in various bulk forms, such as salmon sperm DNA, or bulk-synthesized random DNA can also be used. Competitors may also be unique, such as the use of proteins that are similar to the protein of interest, or non-human proteins that compete with human proteins. With respect to binding, the wash solution can be static or agitated and requires some time for diffusion from the surface into the bulk solution.

The technique eliminates most discrepancies in sample analysis by performing assays and analyzes at collection sites, for example in the toilet under test. Assaying and analyzing fresh urine in a collection vessel (ie, toilet) eliminates the need to store and transport samples, in addition to the associated discrepancies.

This ability to cheaply perform prescribed proteomics at the consumer level will have profound implications for science and healthcare. Proteomics science lags far behind its potential due to the simple lack of a large number of quality samples needed for meaningful analysis. The technique significantly facilitates both collection and analysis of such samples. Good proteomics science provides good medical diagnostic predictions. However, such diagnostic predictions are of little practical in healthcare if patient-derived sample collection and analysis are not easy. Thus, it will provide good proteomics science and the technology will also provide easy use in healthcare science.

Various other properties of the systems and methods according to the technology are described in this disclosure. Properties, functions, and advantages may be achieved independently in the various embodiments of the present disclosure, or may be combined in yet other embodiments, the further details of which may be referred to in the description and drawings below. Understandable.

FIG. 1 is a schematic diagram showing a nitrogen-vacancy center in diamond. FIG. 2 is a schematic diagram of target molecule detection according to an aspect of the present technology. FIG. 3A is a schematic representation of an interaction mechanism that can be used to detect a target molecule, according to aspects of the art. FIG. 3B is a schematic representation of another interaction mechanism that can be used to detect a target molecule, according to aspects of the technique. FIG. 4 is a schematic representation of the electron energy levels at the center of the diamond nitrogen vacancy (NV) and the transitions between these levels according to aspects of the art. FIG. 5 shows an exemplary light absorption and emission spectrum of an NV center according to an aspect of the present technology. FIG. 6 is a schematic view of optical reading and spin polarization of the NV center according to aspects of the present technology. FIG. 7A is a schematic representation of excitation and emission pulses associated with the NV center according to aspects of the art. FIG. 7B is a graph representation of NV center emission intensity comparing intensities as a function of time in the absence and presence of bound target molecules according to aspects of the art. FIG. 8A represents a first exemplary configuration of a device for detecting a target molecule according to an aspect of the present technology. FIG. 8B represents another exemplary configuration of a device for detecting a target molecule according to an aspect of the present technology. FIG. 8C represents yet another exemplary configuration of a device for detecting a target molecule according to an aspect of the present art. FIG. 8D represents yet another exemplary configuration of a device for detecting a target molecule according to an aspect of the present art. FIG. 9 represents yet another exemplary configuration of a device for detecting a target molecule according to an aspect of the present art. FIG. 10 is a flowchart illustrating exemplary steps in a method of detecting a target molecule according to an aspect of the present technology. FIG. 11 is a flow chart illustrating exemplary steps in another method of detecting a target molecule, according to an aspect of the present technology. FIG. 12 is a flow chart illustrating exemplary steps in yet another method of detecting a target molecule, according to aspects of the technique. FIG. 13 is a flow chart illustrating exemplary steps in a method of manufacturing an apparatus for detecting a target molecule, according to an aspect of the present technology.

FIG. 1 is a schematic diagram showing a nitrogen-vacancy center in diamond. In its pure form, the diamond crystal 100 consists only of carbon atoms 101. When the nitrogen atom 102 replaces the carbon atom in the diamond crystal and is placed adjacent to the pore 103 in the crystal, a nitrogen-vacancy center 104 is created.

の方向である。さらに、いずれかの所定の方向において、窒素置換および空孔のオーダーは反転し得、よって、実際には８つの固有のＮＶ中心の構成が存在する。 As can be seen in FIG. 1, since each carbon has four identical binding partners, four crystals in which the axis of the NV center can be located depending on how the pores are arranged in relation to the nitrogen substitution. There is a scientific direction. They are,

Is the direction of. Moreover, in any given direction, the order of nitrogen substitution and vacancies can be reversed, so there are actually eight unique NV center configurations.

The interaction between the NV center and the molecule depends not only on the distance to the NV center, but also on the relative directions of the two. In addition, there is an optical direction with respect to the excited and detected light. All of these considerations must be taken into account when choosing which plane of the diamond crystal will be used as the detection plane.

The nitrogen-vacancy center is desired by introducing nitrogen impurities and vacancies at a predetermined depth and then baking at 1000K to 1300K to arrange the vacancies through diffusion against the nitrogen impurities. It can be embedded in a crystal structure such as diamond at a depth. Nitrogen defects can be injected at the desired depth via nitrogen pulses during chemical vapor deposition (CVD) of the diamond matrix or by ion beam injection after precipitation is complete. Pore can be injected or created via an e- [54], H + [55] or He + ion beam.

Since natural diamond contains about 1% ¹³ C, which interacts with the NV center by having 1/2 the nuclear spin (which is the paramagnetism of the nucleus), the relaxation time of the NV center causes CVD. It can be increased by using to grow an isotope-pure ¹² C diamond deposition layer on an existing crystal and to create NV centers within this layer. Similarly, a 1/2 nuclear spin of ¹⁵ N is preferred over a nuclear spin 1 of ^{the 14 N isotope.} Also, since nitrogen substitutions retain spin, the higher the percentage of nitrogen that can be converted to NV centers, the more transparent the sample is from a magnetic point of view.

Under proper CVD conditions, the orientation of the NV centers produced can be significantly biased. That is, a single orientation can occur in preference to the others, rather than all eight possible orientations being input equally. Higher alignment of 99% is possible. This can be very useful for making devices in which all NV centers can be identical and optimally oriented. For example, if the detection surface of the diamond is the (111) plane and the majority of NV centers can be oriented along the [111] direction, they can all be oriented perpendicular to the detection surface.

In some embodiments of the technique, it is desirable to make a very thin layer of diamond attached to another substrate, such as silicon, in which additional detection electronics are present. Another use for ion implantation is to insert a "break layer" at a particular depth into a thick diamond crystal, for example using a hydrogen atom. After bonding the thick diamond crystal to another substrate, the diamond may be mechanically impacted and will break along the fracture layer, leaving a thin layer of diamond crystal. Similar techniques are already in use in the semiconductor industry.

FIG. 2 is a schematic diagram of target molecule detection according to an aspect of the present technology. The capture reagent 200 is attached to the surface 202 of a substrate 203 (eg, a crystal film, a diamond film, and / or a high-purity insulator such as single crystal diamond) in the immediate vicinity of the color center 204. When the color center is illuminated with excitation light 208 (eg from light source 209), it is stimulated to emit fluorescence 210, which is characterized by spectrum and intensity. The capture reagent 200 is in contact with the sample solution 211 (eg, by exposing the surface 202 to the sample solution) so that the target analyte 206 (also called the target molecule) in the sample solution binds to the capture reagent 200. It forms the complex 212, thereby resulting in and / or altering the interaction 214 with the color center, which results in a detectable change in the nature (eg intensity) of the emitted fluorescence 216. At Interaction 214, the color center can detect changes in the electric or magnetic field upon binding of the target molecule to the capture reagent. The properties of the color center (eg, properties related to magnetic resonance or spin of the color center) can be altered by interaction 214.

The sample solution can be a biological fluid. The capture reagent 200 can be a nucleic acid molecule, an oligonucleotide, an aptamer, SOMAmer, or any other binder configured to bind to a target molecule. In some examples, the capture reagent comprises at least a 5-position modified pyrimidine (ie, a 5-position modified pyrimidine such as uridine or cytidine). In some examples, different types of 5-position modified pyrimidines are attached to the surface. For example, at least one 5-position modified uridine and at least one 5-position modified cytidine can be attached to the surface.

The target analyte can be a protein. Proteins are normally folded polypeptide chains, abnormally folded polypeptide chains, unfolded polypeptide chains, fragments of polypeptide chains that may or may not be normally folded, short polypeptides. Includes polypeptides that incorporate unnatural amino acids, and polypeptides that are post-translationally modified (eg, phosphorylation, glycosylation, disulfide bonds, etc.), or integrated into a protein complex. Target analytes may also contain small molecules found in biological fluids such as metabolites.

3A-3B are schematic representations of two promising interactions of detection techniques, where the color center is NV-centered, the target analyte is a protein, and the capture reagent is an aptamer.

FIG. 3A is a schematic representation of an interaction mechanism that can be used to detect a target molecule using a magnetic spin label according to an aspect of the present technology. Stable organic molecules are usually diamagnetic, and paramagnetic molecules (ie, "free radicals") have unpaired electrons, so they are chemically active and through chemical reactions. Tends to lose spin. Stable free radicals that escape exposure to the biochemical environment are exceptional. Spinlabel is a stable paramagnetic organic molecule or complex capable of binding to other molecules, especially nucleic acids and amino acids, specifically developed for site-specific spinlabels of large biomolecules.

The most commonly used spin labels are metal chelators such as EDTA, which have been developed for incorporation into nucleic acids and complex with nitrogen oxide-containing small organic molecules or paramagnetic metal ions with high affinity. Nitrogen oxide derivatives of nucleobases have also been developed. In recent years, triarylmethyl (trityl) radical derivatives have also become popular for labeling both proteins and nucleic acids. Both EDTA derivatives of deoxyribo-thymine and deoxyribocytosine have been developed in the form of phosphoramidite for use in DNA synthesizers. In measurements where the spin label is intended to simply change the relaxation time of the NV center, it is important that there is a magnetic interaction and that a wide variety of spin labels are applicable. However, in measurements where the spin label of the label is presented directly, the individual spectra of the spin label become relevant. For example, in DEER, the narrower the spectrum and the longer the spin label life, the better. As a specific example, ^{triplet splitting of the spectrum is disadvantageous due to the ultrafine coupling to 14} N in DEER, and a single-line spectrum of deuterated trityl is the preferred choice.

In FIG. 3A, the aptamer 301 coupled to the magnetic spin label 300 is attached to the detection surface 202 of the diamond crystal 100 in the immediate vicinity of the color center 104. The target protein 302 in the sample solution binds to the aptamer to form a complex 303, by altering the proximal relationship between the spin label and the spin label, which affects the interaction 304 between the spin label and the NV center. It results in a detectable change in the fluorescence properties of the NV center.

More specifically, FIG. 3A illustrates a particular configuration designed to maximize magnetic interaction by placing a spin-labeled aptamer near the NV center in the diamond. Three-dimensional structural changes during protein binding can move the spin label in relation to the NV center. Movement closer to the NV center increases the magnetic field at the NV center, resulting in measurable changes in the dynamic or quasi-static features of the lower level spectrum of the NV center.

The lower level spectrum can be probed using a detector assembly configured to illuminate the NV center with excitation light and detect the emission of electromagnetic radiation from the NV center. The detector assembly detects changes in the lower level spectrum by illuminating the NV center with microwave radiation over a frequency range that includes the lower level resonant frequencies and detecting changes in the radiation emitted by the NV center. Can be done. Radiation at the resonant frequency can induce the conversion of NV-centered ground-state electrons from the first lower level to the second lower level, thereby inducing changes in the characteristics of the emitted radiation. One example of the characteristics of emitted radiation is the relationship between the frequency of the excitation light and the frequency of the emitted light. In addition, the addition of spin labels with microwave excitation opens up a wide range of options for manipulating spin label interactions with NV centers.

FIG. 3B is a schematic representation of another interaction mechanism that can be used to detect a target molecule using electrostatic interactions, according to aspects of the art. The aptamer 301 is attached to the detection surface 202 of the diamond crystal 100 in the immediate vicinity of the NV center 104. Both cations 308 and anions 309 are present in the fluid being analyzed. The negatively charged skeleton of the aptamer attracts cation 308. These bound ions affect a local electric field relative to the NV center so that when the NV center is irradiated with excitation light 208, it emits a fluorescence 310 characterized by spectrum and intensity. Be stimulated. The protein in the sample solution also has ions bound to the protein by its charge. This protein binds to aptamers to form a complex 303, thereby altering the charge distribution and thus the local electric field relative to the NV center, resulting in a detectable change in the properties of the emitted fluorescence 312. ..

More specifically, FIG. 3B illustrates a configuration in which electrostatic interactions can be explored. The presence of mobile charges near the surface significantly affects the relaxation time at the NV center. There is also a negative charge bound to the NV center itself and a negative charge bound to the skeleton of the aptamer. In addition, the target protein itself can be charged. These charges are screened by counterions in solution. The binding of protein molecules to aptamers rearranges this charge distribution with respect to the NV center. In addition, the protein molecule itself has a significantly lower dielectric constant than water, and its presence affects the screening of charge distribution. Both actions result in quasi-static and dynamic changes in the electric field that reside in the NV center, affecting the characteristics of the lower-level spectrum.

The degree to which static or dynamic changes in the lower level spectrum dominate the interaction can be influenced by the environment in which the measurements are made. If measurements are taken at room temperature, mobility of charges in the solution and molecules themselves, it is expected to contribute to significant dynamic changes that may be characterized by direct measurement of T _1. In contrast, when measurements are taken at temperatures below the freezing point of the biofluid, these dynamic changes are suppressed, allowing direct detection of static division at lower levels. For spin-labeled aptamers, measurements in the frozen state significantly increase the relaxation time of the spin label and are used to directly measure changes in the distance between the spin label and the NV center. It enables technologies such as DEER (elector-elector double resonance).

The optimal configuration for positioning the NV center also depends on whether the spectral changes are dominated by quasi-static or dynamic changes. If the dynamic change is dominant, and a direct T ₁ measurements are used, NV center is located at a position closer to the surface, and there is a possibility that may be positioned closer together. On the other hand, careful measurement of quasi-static changes to the spectrum requires a longer relaxation time of high fidelity (first T ₁ ), thus being further away from the surface and well separated from each other. Requires NV center. The distance of the NV center from the surface is between maximizing the interaction between the aptamer and its binding target and maintaining a long relaxation time to create a quasi-static electric and magnetic field NV sensitive detector. It is a compromise of.

Figures 3A-3B study two limitations of interaction, where magnetic and electric fields are the dominant ones that result in changes in the NV center upon coupling of the analyte. As when the SELEX process was modified to screen for slow dissociation of SOMAmer production, the candidate aptamers were bound to devices such as those described herein and NV-centric to target binding. Steps for measuring response may be added to subsequent selection rounds. In this method, the SOMAmer that maximizes the contrast of the signal during the binding event can be maximized.

FIG. 4 is a schematic representation of the electron energy levels at the center of nitrogen vacancy (NV) in diamond and the transitions between these levels according to aspects of the art. More specifically, FIG. 4 is a simplified outline of the electron energy levels of negatively charged NV centers in diamond and the transitions between these levels. The ground state 399 of an electron at the NV center contains three lower levels (also called substates or states). The lower level 400 of zero spin has the least energy and ^{is represented as | 3} A ₂ , 0>. The lower levels 402 of the spins of m _s = + 1 and m _s = -1 have the same energy in the absence of an external field ^{and are represented as | 3} A ₂ , ± 1>.

External field, _{m s} = affects the spin state of ± _1, without affecting the spin state of the _m s = _0, to lower the energy of the spin state of the m s = _-1, the _m s = + 1 Increases the energy of the spin state. For example, a magnetic field of 1027 Gauss arranged along the axis of symmetry of the NV center ^{reduces the level of | 3} A ₂ , -1> to the | ³ A ₂ , 0> level at room temperature. The presence of a magnetic spin label in the immediate vicinity of the NV center has a similar but significantly smaller effect. Irradiation with a microwave at a modified frequency of 404 (eg, resonance frequency) _{induces a transition from a spin state of basal ms} = 0 to a spin state of basal _ms = ± 1. | ³ E, denoted 0>, the transition to the spin state 406 _m s = 0 from spin excited in _m s = 0 of the base or, ^| 3 E, denoted ± 1>, base the m _{s =} transition from spin state of ± 1 to the excited m _{s =} spin state of ± 1 408 may be induced by irradiation with excitation light correction frequency 208.

The transition from the excited state to the corresponding ground state is achieved by the emission of red photons 418. Alternatively, the so-called "dark transition" is achieved because most or all of the transitions from the excited state to the ground state are achieved with no photon emission or only with longer wavelength infrared photon emission 412. It can occur via "transition)" 410. The dark transition 410 can be achieved with the emission of photons 412 in the infrared range absorbed by the diamond lattice, or along a path 414 with no photon emission. In most cases, the dark ^{transitions, |} bring spin transition 416 to _{^{3 A 2, 0> | 3}} E, from ± 1. | 3 ^E, about 30% of the ± 1> level transition on the electron transitions to the ground state via the dark transition, resulting in detectable photon emission of. Therefore, the electrons in the spin state of _{m s} = ± 1 bring about only about 70% of the fluorescence of the electrons in the spin state of _{m s = 0.}

More specifically, FIG. 4 shows a schematic energy level structure consistent with the findings and calculations for negatively charged NV centers. Six electrons are located in the ground state "molecular orbital" of _{C3V symmetry (imposed by a static lattice structure).} These orbitals are a linear combination of four orbitals that are consistent with the calculation of the diamond band structure. ^{Energetic, this ground state, labeled 3} A _{2 in} FIG. 3, is a deep level state in the bandgap. ³ a first excited state to be E and the label is 1.94eV above basal state. If not so localized, it is further away from the conductor by more than 1 eV. The difference of 1.94 eV between the two band states corresponds to the observed 638 nm zero-phonon absorption and emission lines shown in FIG.

Both the ground state and the excited state are spin triplets with S = 1, but from now on, Applicants will focus only on the lower levels of magnetism in the ground state. Therefore than using the description of unmanageable state in FIG. 3, the present applicants have simply often refers to the state of the m s _{= 0} or m _{s =} ± 1,, which is the applicants Is understood to refer to the ground state.

If there is no applicable place, the degeneracy of the _m s = 0 and _{m s} = ± 1 in the lower level in the ground state, a large null field along the NV center axis splitting (zero field splitting) by (2.88GHz) First enhanced, it reflects the strongly non-spherical wavefunction of the six electrons. In the presence of the applied magnetic field and / or electric field, _{the lower levels of m s} = 0 and m _s = ± 1 are further split. In a field parallel to the NV central axis, the split is 2.8 MHz per gauss in a magnetic field and 3.5 MHz per V / m in an electric field. This drawing does not represent the energy difference at scale, and the lower level splits of magnetism are about five orders of magnitude smaller than the major splits (1.95 eV) between the ground and excited states.

The transition between the lower levels 400 and 402 of the ground state and the excited states 406 and 408 is visible and results in an interesting photoluminescence property of the NV center. FIG. 5 represents an exemplary spectrum of light absorption and emission of NV centers at room temperature according to aspects of the art. The absorption spectrum 500 peaks at about 570 nm and shows how efficiently photons of a given wavelength are absorbed by the NV center. Emission spectrum 502 peaks at about 690 nm and indicates the relative intensity of light emitted at a given wavelength. Absorption of photons of any wavelength in the absorption spectrum can result in emission of photons of any wavelength in the emission spectrum. The expected diffusion of the spectrum from another wavelength is due to the dissipation of the vibrational energy into the molecular lattice or the absorption of thermal energy from the molecular lattice. Both spectra share a peak 504 in which the absorbed and emitted photons have the same energy. This peak is called the zero-phonon line, where these spectra converge as the atmospheric temperature reaches absolute zero and the vibrations of the lattice are frozen.

More specifically, both the absorption and emission spectra show a strong absorption coefficient, a short (about 4 ns) lifetime with respect to the excited state, and a wide phonon spread resulting in substantially non-fading strong fluorescence. Route direct absorption and release, angular momentum of the S and m _s remain to both uncharged the (absorbed photons or emitted photons may be offset by the momentum of change in trajectory "molecular" ). In contrast, strong phonon coupling allows for another decay path (via the intersection of intermediate systems), which _{alters ms} by one, as shown on the right side of FIG. This decay path goes through multiple intermediate states, and any photon energy emitted is further in the infrared compared to the photons brought about by the direct path. Thus, this is often referred to as the "dark path" and is higher for electrons excited from the lower level of _{m s} = ± 1 than those excited from the lower level of _{m s = 0.} Adopted by possibility. The net result for electrons excited from the ground state _ms = 0 lower level is higher visible fluorescence intensity compared to those excited from the _{ground state ms = ± 1 lower level.}

FIG. 6 is a schematic view of optical reading and spin polarization of the NV center according to aspects of the present technology. The excitation pulse 600 irradiates the nitrogen-vacancy center and stimulates the emission of the pulse 602. The initial intensity 604 of the emission pulse, I _start, is the smallest point of the pulse and represents the relative set of states _{m s} = 0 and m _{s = ± 1 at the start of the measurement.} This strength, when the spin state is polarization relative state of _m s = 0, rapidly rises to the maximum intensity _{606, I max.}

Change of the set of magnetic lower level is exactly is what is observed through the electron spin resonance (EPR), the frequency of the excitation of the microwave applied here is consistent with the division of the lower level of the m _s. Fluorescence intensity depends on the lower level set described above and allows optical detection of ground state magnetic resonance (ODMR) on the NV center. Applicants continue to refer to ODMR detection, but note that direct electrical detection of photocurrents at the NV center is illustrated and can act as another detection mechanism.

Traditional magnetic resonance applies a continuous or pulsed microwave / RF electromagnetic field to simultaneously induce transitions between the magnetic sublevels of ^{multiple (> 10 13) spins or mix these sublevels.} That is, it operates with a temperature-dependent macroscopic (ensemble average) magnetization. At zero absolute temperature, all spins are at their lowest energy lower level and the magnetization of this system is maximum, so this state is 100% spin-polarized. At finite temperature, the lower level set decreases the ensemble average polarization as the temperature rises, according to Boltzmann statistics. At room temperature, the energy difference between the lower levels is so small that the sets of lower levels of different magnetism are roughly equal, resulting in a polarization of only 0.1%.

The magnetization generated from the difference in the lower level set is called longitudinal magnetization, or is called the z component of magnetization because the conventional Z axis is selected parallel to the quantization axis. At either temperature, the longitudinal magnetization has an equilibrium value proportional to the spin polarization. The transverse magnetization, which is the XY component of the magnetization, is the ensemble average of the mixed state (the eigenstate is located along the Z direction). Its equilibrium value is zero.

Lower-level transitions or mixed states (with a non-zero ensemble average of transient transverse magnetization) can be created by applying an RF / microwave electromagnetic field with photon energies consistent with the lower-level splitting energy E. .. The corresponding frequency is defined by E = hf _L (where h is Planck's constant and f _L is known as the Larmor frequency). The mixed state (and thus the ensemble average transverse magnetization) precesses (rotates) at the Larmor frequency. Usually, the EMF induced by this rotating macroscopic magnetization is detected via a coil or a resonator.

After the creation of the mixed state, the signal of transverse magnetization decays over time in principle for two reasons:
1. 1. Each spin, experienced a place for different field or changes during the precession, i.e., the energy difference between the lower level m _s are different, whereby the frequency of precession of the mixed state are slightly different .. While they initiate precession in phase, the phase difference is constructed by the frequency difference of the precession, and the ensemble average (total vector) transverse magnetization progresses to zero. Since the macroscopic magnetic moment that precesses collapses to zero, the inevitably induced signal also collapses. The characteristic time of this decay caused by the loss of "phase coherence" _{is called T 2} or lateral relaxation time. In more detailed experiments, decay due to static field differences (ie, magnetic field inhomogeneity across the sample) is eliminated, and decay times that reflect only dynamic changes during the measurement are therefore phase memory. time) is referred to as _{T M.} Loss of transverse magnetization in this form does not require energy exchange with the environment.
2. At finite temperatures, spins can exchange energy with the environment and flip from one sublevel to another, i.e., eigenstates have a finite lifetime due to their interaction with the environment. More precisely, the fluid (time-dependent) electromagnetic field provided by the environment needs to have a non-zero Larmor frequency field component in the lateral direction to induce a change in state. As a result of these changes, the precessing transverse magnetization is lost as the magnetization returns in the z (or longitudinal) direction. Characteristic time associated with this process (the time required to return to thermal equilibrium set distribution eigenstates) is called T ₁ or longitudinal relaxation, or it requires a energy exchange with the environment, spin -Called the lattice relaxation time. This process imposes an upper limit on the time of any precession collapse, such as _{T 2} and T _m.

Unlike conventional EPR detection, ODMR detects longitudinal magnetization. However, the lateral and loss of its benefits are significantly complemented by a significant increase in susceptibility: low-energy magnetic low-level transitions with near-perfect quantum efficiency and photons with energies greater than 1,000,000 times. Can be observed through.

In addition to allowing ODMR detection, in isolated NV centers, dependent fluorescence m _s also allows the operation of a set of lower level. This is because the lower level lifetime of magnetism is much longer than the lifetime of fluorescence. At sufficiently high excitation intensities, the fluorescence rate is limited only by the lifetime of the fluorescence and can be 100 MHz or higher. For example, _{the probability that excitation from the lower level of ground state m s} = ± 1 will retain its quantum number w = 0.7 and the probability of returning to the lower level of _{m s = 0 via the dark path 0.3.} The probability of finding any of the spins in the m _s = 0 state after 10 cycles is 98%, i.e. 98% spin polarization can be constructed for a macroscopic sample. Since the NV-centered T ₁ is usually much longer than 100 microseconds, a sufficiently strong 100 ns pulse can create spin polarization equivalent to cooling the diamond from room temperature to 0.3 K. Initiating any magnetic resonance measurement with nearly completely polarized spin is a 100-fold increase in signal compared to starting with equilibrium spin polarization at room temperature.

The same pulse that polarizes the set of spins also acts as a measure of spin polarization, as shown in FIG. Maximum fluorescence occurs when the sample is polarized at 100% spin at the lower level of _{ms = 0.} Applicants define the maximum fluorescence intensity as I _max. Assuming that Applicants use the probabilities from the above example and that Applicants start in thermal equilibrium, the fluorescence response when the excitation light is turned on at t = 0. Is fluorescing at maximum efficiency _{1/3 of the lower level set of m s} = 0, and fluorescing at 70% efficiency (1/3 + 2/3 * 0.7 = 0.8) m _s = ± It is 80% of _{I max} , which corresponds to 2/3 of the state of 1. This strength is a ratio which is determined by the intensity of the excitation light reaches the asymptotic to I _max.

として、最初の蛍光強度Ｉ_{ｓｔａｒｔ}の観点から表され得る。 In this case, the first polarization at the lower level of _{m s = 0, P 0,} is

_Can be expressed in terms of the initial fluorescence intensity I start.

In the case of w = 0.7 analyzed above, when I _start = I _max , P ₀ = 1. When I _start = 0.7 * I _max , P ₀ = 0. When I _start = 0.8 * I _max , P ₀ = 1/3, and so on.

The unique properties of NV centers summarized above mean that it is easy to construct experimental settings that can detect fluorescence of a single NV center using a commercially available fluorescence microscope. Since NV centers can be separated by hundreds of nanometers, the fluorescence of a single NV center observed under a microscope contains all the spectroscopic information at the lower levels of magnetism in a single spin. When a single spin is observed, there is no ensemble average as in the case of conventional EPR measurements, but there is still a direct correspondence between the ensemble average and the time average of a single spin. To relate these two, the ensemble average polarization value needs to be replaced with a lower level probability, i.e., an optical that results in _{98% of the macroscopic ensemble's spin polarization of m s = 0.} The excitation pulse is transformed into finding a single NV center with _{a probability of m s} = 0 with a 98% probability. Thanks to high fluorescence, rapid repetition and sufficient time averaging, reasonably short experimental times can be achieved with few statistical errors.

The nature of the NV center is well suited for time domain magnetic resonance: 100% polarized initial state, detection of sensitivity of a single center, and long relaxation time, more sophisticated high resolution spectroscopy. The fact that all time domain rf / microwave pulse sequences ever invented for this can be applied makes a single NV center the most sensitive quantum measurement tool available.

FIG 7A~7B is relaxation time via a fully optical measurement, represents the means for measuring the T _1. The lower level of spin of m _{s = ± 1 emits at a fluorescence intensity of about 70% of the lower level of m s} = 0, so that when evenly divided, the total fluorescence intensity is m _s = spin. It is about 80% of the case where it is completely polarized to the lower level of 0. A series of pulses, such as in FIG. 6, are used to evaluate the relative set of lower levels of _{m s} = 0 and m _s = ± 1 and to repolarize the spin to the lower level _{of m s = 0.} Allows measurements to be made.

FIG. 7A is a schematic representation of the optical excitation pulse 708 and emission pulse 710 associated with the NV center according to aspects of the present technology. NV centers in diamond are each sufficiently long period to effect complete polarization of NV centers to a lower level of m _{s =} 0, it is illuminated by a series of excitation light pulses. _{The spacing τ i} between these excitation pulses varies. Fluorescence intensity immediately after the start of the excitation pulse (painted rectangle _{(filled square) I 0, I} 1 ...) is taken if the spin state should be fully polarized at a lower level of _m s = 0 , Reference intensities (open rectangles), as well as measured.

FIG. 7B is a graphical representation of the emission intensity of NV centers comparing the intensity as a function of time in the absence and presence of bound target molecules according to aspects of the art. More specifically, the difference between the first intensity and the reference intensity in FIG. 7A, is plotted as a function of the time interval tau _i between them, how until lower levels return to thermal equilibrium Indicates if time is needed. The exponential fit of intensity = I ₀ * exp (−τ / T ₁ ) results in a direct measurement of _{T 1.} Data point 700 shows a quicker return to equilibrium, so these exponential fits 702 result in a _{shorter T 1 than data points 704 and their exponential fit 706.} Different value of T ₁ occurs as a result of or unbound analyte is bound to the aptamer.

More specifically, in FIGS. 7A-7B, a series of excitation pulses of sufficient duration and intensity to optically depolarize the NV center are applied with various waiting times between them. This excitation wavelength is shorter than the zero-phonon line, and in practice 532 nm is often used. At the same time that the excitation pulse is applied, the emission from the NV center is monitored with a bandwidth that includes part or all of the emission spectrum 502.

First, the ground state is thermally equilibrate, thus the first level I ₀ of fluorescence, because the state of the m s ₌ ± 1 and m _{s =} 0 is present substantially equal, less than the maximum possible I _m Corresponds to the level. Over the course of the first pulse, release, as the state of the m s _{= 0} will be present by dynamic spin polarization process described above, grow to the maximum value saturates at I _m. After the wait time, a second pulse is applied. If the waiting time is sufficiently short, the interaction with the surrounding environment does not have enough time for balancing the population of ground state, m s _{= 0} is still many exist, therefore the measured initial release intensity _{I 1} is still close to the maximum value _{I m.}

Spin polarization is again occurs during the remainder of the second pulse, this state is polarized to the intensity of the re-m _{s =} 0 and the corresponding I _m. The longer wait time then follows and the entire process is repeated again. Since this process is repeated as a result, the waiting time is substantially longer than the relaxation time of the NV center, the ground state is thermally balanced, and all three lower levels are equally present. , The first emission measured is again equal to _{I 0.}

_{By plotting the initial emission intensities I 1} , I ₂ , I ₃ ... As a function of waiting time, a graph such as that shown in FIG. 7B can be created. Fit to the graph may result in a relaxation time T ₁ of the NV centers. 7B illustrate the adaptation thereof corresponding to the example and different value of T ₁ of the two data sets. This measurement sequence is very similar to that used in standard pulsed magnetic EPR experiments, where the initial state is the inverted thermal equilibrium magnetization-M _{0 prepared by the microwave π pulse.} The late time value is read by detecting the microwave emission initiated by another microwave π / 2 pulse. However, in this case, no magnetic or microwave antenna or resonator is required, which greatly simplifies the measurement.

In addition to microwaves, more detailed measurement techniques will be possible. For example, if a fully optical T ₁ measurements described above, is oriented perpendicular to the axis of symmetry of the frequency and NV centers of π pulse (zero field splitting of the added microwave immediately after all polarization optical pulses (With a field) provides the first population of zeros for the _{m s = 0 state.} It was measured not measured and without involving pulse microwave alternately, by their differences plots and adapted, capable of measuring T ₁ without requiring adaptation of the values of the remaining (thermal equilibrium) signal. This, m _{s =} + / in the absence of an external magnetic field - the first state is degenerate (equal energy) for, it should be noted that the act very simple.

Adding an external field further increases the possibility of measurement. If an external static field _{H 0} is _imposed, m s = + / - energy level of the first state divide, thus _m from _m s = 0 s = + 1 and _{m s} = from _m s = -1 The transition to responds to the excitation of microwaves of different frequencies. As long as the applied field is small or parallel to the axis of symmetry of the NV center, the optical response does not change and can continue to be used for reading. The optical polarization in the m _s = 0 state continues to act as an ideal starting point for the experiment, and the first fluorescence intensity measured during the optical excitation pulse at a later time is _ms = 0. It can be used to measure a lower level population of 0, i.e., it can act as a sensitive longitudinal detection. The time interval between initial polarization and detection can vary and can be used to perform lower level spectroscopy between levels selected by the frequency of the microwave pulse. Note that the external electric field can also split the lower levels and be used more than the external magnetic field.

All known microwave pulse sequences in the range from a simple Hahn echo, CPMG (Carr-Purcell-Meiboom-Gill) to MREV-8, search for NV-centric coherence time, i.e. at millisecond time intervals. It can be used to determine small field variability up to a few tenths of a milligauss. Prerequisites for these measurements is a long T _1, away from the surface (15-20 nm) NV center well, freeze aptamers, proteins, and the movement of ions, to freeze the liquid that is being tested Need that.

In addition, the change in distance between the spin label attached to the aptamer and the NV center induced by protein capture can be measured in a DEER (Double Electron-Electron Response) experiment. Applying a second microwave excitation at different frequencies to reverse the spin on the spin label while reversing the lower level precession of the NV, increasing the frequency difference of the precession due to spin reversal. It can be detected with accuracy and enables measurement of distances with an accuracy of up to 20 nm and nanometers or less. In order to perform DEER over the required distance, the relaxation time of the NV center and spin label on the aptamer needs to be in the order of milliseconds and it is necessary to freeze the liquid to be tested again. ..

As mentioned above, the lateral separation between NV centers may be large, for example above 100 nm, to prevent significant interactions between NV centers. However, aptamers are small, in the order of a few nanometers, so Applicants distinguish between two different perceptions. In the first recognition, aptamers are densely bound to the surface so that any given NV center interacts with multiple aptamer sites randomly distributed within the interaction of the NV center range. Thus, mean interaction changes are measured across multiple sites.

In the second possibility, site-specific surface chemistry is used to ensure a 1: 1 pair between the aptamer site and the underlying NV center. This can be achieved by using surface chemistry that is affected by the charge present at the NV center or by fluorescence at the NV center. This configuration has the advantage of providing nearly identical measurement sites that provide the sensitivity of a single protein and the property of distinguishing between the exact target protein and the non-specific binder. This ability to collect statistics based on a molecular yes or no decision means that the second configuration can provide excellent resolution and dynamic range, given the same number of NV centers.

One of the purposes of the present technique is to measure the concentration of a plurality of proteins at the same time by using an aptamer specific for each individual protein. There are at least two fundamentally different ways in which this can be achieved.

In the first possible detection paradigm, there are numerous separate NV centers and aptamer sites, each measured by an independent detector, for any given protein target. It efficiently performs a number of single NV center experiments. If there is an additional 1: 1 pairing between the NV center and the aptamer site, the number of bound proteins can be counted to yield a singular that represents the concentration of the specific target protein.

In the second possible detection paradigm, multiple NV centers and aptamer sites for a given protein target are measured simultaneously by a single detector. This abandons the advantage of molecular identification and requires the creation of a calibration curve to quantify the concentration of the target protein, which can reduce the complexity of the equipment required.

In each case, ideally all NV centers are excited and detected at the same time, however, it uses multiple measurement protocols to address the fact that different aptamers can have different responses. Do not exclude.

In both cases, different parts of the diamond (or more generally crystal) surface are specialized for measuring the concentration of different proteins. However, there are differences in the number of detectors required. For example, to detect the concentration of 10,000 protein, in the first case may require 10 about ^eight detectors, but in the second case requires only slight 10,000 detector And.

8A-D represent different configurations for probing biochips based on the present technology. For the same NV center located along the [111] direction near the diamond surface ending at (111), all measured NV centers are equivalent and most sensitive detection regions are true of the NV center. Due to its location on top, it is an ideal configuration, but other constraints, such as which orientation of diamond crystals can be easily produced, may not make this possible. Some device configurations allow this situation more than others.

FIG. 8A represents a first exemplary configuration of a device for detecting a target molecule according to an aspect of the present technology. In the simplest configuration, the aptamer 301 is attached to the surface of the diamond crystal 100 located in the immediate vicinity of the nitrogen-vacancy center 104 located at a predetermined depth below the detection surface 202. The nature of the fluorescent synchrotron radiation 418 induced or stimulated by the excitation light 208 is measured to detect the presence of the protein-aptamer complex 800. The presence of the protein induces a change in one or more properties of the nitrogen-vacancy center, which change is detected based on fluorescence radiation from the nitrogen-vacancy center.

Configuration shown in Figure 8A, is all that is required to perform all of the optical T ₁ measurements described above. This imposes the least stringent conditions for NV-centric equivalence. A (100) diamond surface with all eight different possible NV center orientations can be used. In this case, the axis of symmetry of the NV center is not perpendicular to this surface, which is not ideal, but all eight orientations are offset by the same amount, so the NV center functions equally. One of the drawbacks is that the details of the dipole-dipole interaction minimize the magnetic interaction directly above the NV center, so most sensitive interaction regions are lateral from each NV center. It is to be placed some distance in the direction. This does not apply to electrical interactions. This orientation is also not ideal for optical excitation and reading using light that is internally reflected inside the diamond. However, this represents an appropriate compromise between the two, allowing the use of diamond surfaces, which are easier to manufacture.

FIG. 8B is another exemplary configuration of a device for detecting a target molecule according to an aspect of the present technology. A microwave source 806 is added to the configuration represented by (A), in which the microwave emission 802 is directed to the diamond surface by the microwave antenna 804. The microwave source is consistent with the zero electrolytic splitting frequency (2.89 GHz) and is in the direction of ensuring that the microwave magnetic field angles are the same for all NV centers, as shown in FIG. 8B. It can also help with the accuracy of the measurement. The intensity of the fluorescent synchrotron radiation 418 may be measured and the behavior of the color center resonance within the diamond can be identified based on the relationship between the measured intensity and the frequency of the microwave emission 802.

FIG. 8C represents yet another exemplary configuration of a device for detecting a target molecule according to an aspect of the present art. A magnetic field supply source 810 in which an external magnetic field 812 is applied to the diamond surface is added to the configuration represented by (B). _{The addition of an external static field H 0} as described on FIG. 8C may be required for the measurement of phase coherence (T _2).

FIG. 8D represents yet another exemplary configuration of a device for detecting a target molecule according to an aspect of the present art. A second microwave supply source 808 that shares the same microwave antenna 804 as the first microwave supply source 806 is added to the configuration represented by (C). Second (independent frequencies) microwave source shown in Figure 8D may be required spin label for performing DEER experiment - magnetic fields of all of the microwave is ideally against H ₀ Note that it is almost vertical. Ideally, this configuration uses a (111) surface with NV centers arranged along the [111] direction described above, as only NV centers parallel to the field can be optically read at high magnetic fields. do. If all NV center orientations are applied equally, 75% of them cannot be used. The optical excitation and fluorescence emission is ideally approximately parallel to the NV axis.

For non-equivalent NV centers (such as crystals ending at (100) where all eight input have different orientations), a possible solution is a field that is properly aligned for these. All of these are processed in succession by using only 1/8 of the NV center in and applying different field orientations and microwave frequencies. Another possible solution is to use what is known as "field cycling" in a resonant magnetic field. In this case, a large field (eg, more than twice as large as zero electrolytic splitting) can be applied along the [100] axis during microwave operation and switched to zero during optical polarization and detection.

FIG. 9 represents yet another exemplary configuration of device 899 for detecting a target molecule, according to aspects of the technique. More specifically, FIG. 9 is a schematic (not on scale) of the biochip integrated under the present invention. This figure represents one embodiment of a detection technique utilizing spin labels, aptamers, and nitrogen-vacancy centers, but other embodiments of detection techniques are possible. The biochip is composed of four layers: a capture layer 900, a diamond layer 902, a filter layer 904, and an integrated detection and treatment layer 906. The capture layer 900 comprises a channel that directs a sample solution containing the target molecule 206 (eg, target protein 302) of interest through the surface of the diamond layer on which the SOMAmer 301 bound to the magnetic spin label 300 is attached. Will be done.

The mechanism in the capture layer is the region of the detection surface of the diamond layer on which the same SOMAmer is attached. Each mechanism contains one or more SOMAmers specific for separate protein analytes, and x-to the combined assembly of NV centers in the diamond layer and the individual photodetectors in the integrated detection and treatment layer. Corresponds with the y coordinate (Fig. 9).

Each SOMAmer 301 or other capture reagent 200 can be a capture reagent of a particular type or species configured to bind to a target molecule 206 of a particular type or species. The capture reagent seed can be attached to the functional groups of the capture reagent. The capture layer 900 may include capture reagents 200 belonging to at least two different species, whereby at least two target molecules 206 can be detected by a biochip device. In some embodiments, the capture layer 900 comprises a large number of species, such as hundreds, thousands, or more capture reagents.

Some preferred embodiments of the invention do not include spin label. Binding of the protein molecule to the SOMAmer molecule results in a change in the interaction 304 between the SOMAmer and the proximal nitrogen-vacancy center 104, such change being detected via fluorescence emission 216. Surface chemistry and SOMAmer are optimized to result in large relative differences in interaction during binding of protein targets to aptamers.

The diamond layer is composed of high-purity diamond produced by chemical vapor deposition, in which the layer of the NV center 104 is embedded at a predetermined fixed distance 105 from the surface. In some examples, a given fixed distance, which can also be expressed as depth, ranges from 5 to 20 nanometers, or 15 to 20 nanometers. The NV centers within the diamond layer are separated by distances greater than a predetermined fixed depth, ensuring that the interaction between them is small compared to the interaction with the molecules on the surface. The thickness of the NV center layer is small compared to a predetermined fixed depth. The distance between the nitrogen-vacancy center and the SOMAmer is chosen so that changes in the interaction during protein binding to the SOMAmer are possible and / or small enough to be detected. An external magnetic field 812 can be present. Diamond layer may comprise ^{100% 12} C diamond.

The excitation light 208 (provided by a light source such as a laser) stimulates fluorescence at the NV center. External microwave 802 transitions ground-state electrons between these lower levels. The filter layer is composed of a layered dielectric to form a dichroic bandpass filter 912, which is adhered to a diamond layer 902 on one side and an integrated detection and treatment layer 906 on the other side. In addition to passing radiated light from the NV center while blocking all other wavelengths, the filter layer 904 also blocks radiated light outside the normal narrow range of angles, thereby blocking adjacent NVs. Minimize crosstalk in center-to-center detection.

The filter layer is designed to be a narrow bandpass filter per emission wavelength (680 nm) at the nitrogen-vacancy center. In addition, the filter layer blocks light that is normally outside the narrow band angle to minimize crosstalk between adjacent mechanisms. The filter layer also acts as a mechanical bond between the diamond layer and the integrated detection and treatment layer. An optically transparent and mechanically rigorous adhesive can be used to bond the filter layer to the diamond layer on the top surface and to bond the filter layer to the integrated detection and treatment layer at the bottom. The filter layer is constructed from a layer of dielectric that relies on the principle of wave interference to block light of a particular wavelength. The design and manufacture of filter layers is outside the scope of this technology.

The integrated detection and processing layer 906 consists of a CMOS avalanche photodetector 914 driving a high speed electronic gate 916 driving an electronic event counter 918. The photodetector 914 may also be referred to as a detector assembly. In the present technology, the detector assembly may be configured to illuminate the color center with excitation light of one or more frequencies and detect the emission of electromagnetic radiation from the color center. The output of the event counter is collected for analysis. The designated coordinates 920 are selected so that the x and y coordinates are on the surface surface of the upper diamond layer and the z coordinate is orthogonal to this surface. The wavelength in the range emitted by the light source (ie, the spectrum of excitation light 208) may or may not be the same as the wavelength in the range detected by photodetector 914. The wavelength range of the excitation light may or may not partially overlap the wavelengths in the range configured for detection by the photodetector 914.

The integrated detection and processing layer may be designed for lithographic production and includes both electronic components and the connections between them. The integrated detection and processing layer may be made using lithography and may be adhered to the filter layer (or diamond layer if the filter layer is excluded) using an optically transparent adhesive. .. The integrated detection and processing layer may consist of a photodetector layer, a gating layer, an AD (analog-digital) layer, and a bus for moving the stored data to the processor. Although FIG. 9 shows these layers distributed in the z direction, such an arrangement is not necessary.

The photodetector layer may include an array of avalanche photodiodes, each photodiode specialized for a single mechanism on the capture layer and associated with this mechanism in x and y coordinates. Each photodiode may be associated with a fast electron gate in the gating layer. Changes in fluorescence from the NV center can be on a microsecond or less scale, so it is necessary to collect light only in a very short defined time frame to monitor these changes in intensity. Each gate is now associated with an event counter in the event counter layer, which actually makes photon measurements. The data from each event counter is delivered to the processor for data analysis.

There are at least three examples of arrangements in which the capture layer mechanism of FIG. 9 (ie, the region of the detection surface of the diamond layer to which the SOMAmer is attached) can be constructed, each offering different advantages. In the first example, a single SOMAmer is associated with a single NV center, which in turn is associated with a single photodetector. Such a setting provides advantages in terms of detection, but requires precise control of NV-centric or single SOMAmer placement. The main advantage of this form of construction is that it provides individual-based molecular detection, which is a third dimension of specificity (binding) based on the binding characteristics that would be lost in ensemble detection. Allows after affinity and washing). In addition, the target molecules can be measured one at a time, eliminating some calibration steps. The main drawback of this form is that a number of such mechanisms are required to distinguish between different concentrations. Separating orders of magnitude difference in concentration requires hundreds or thousands of such characteristic mechanisms (based on a given level of resolution) for a single analyte, increasing the complexity of the instrument. Can be done.

A second exemplary form of construct relates to a set of multiple NV centers and thus includes a set of multiple SOMAmers of the same type associated with a single photodetector. The random distribution of NV centers in the xy plane (as defined in FIG. 9) and the random distribution of SOMAmer in the set of mechanisms yielded an averaged signal at the photodetector, which is more It should be stable. Concentrations in a range can also be quantified by multiple photodetectors with a single photodetector. However, the use of such an aggregate instead of a single SOMAmer or NV center hinders the specificity of the third dimension made possible by the first form and NV to the concentration of the target protein in the fluid. It is necessary to create a calibration curve to correlate the response of the center.

A third form of construct is a hybrid of the first and second forms, associated with a single NV center, which in turn relates to a single photodetector, multiple SOMAmers of the same type. Contains a set of. Similar to the second form, a range of concentrations can be quantified by multiple SOMAmers with a single photodetector, which also yields an averaged signal. The use of a single NV center can provide a clearer signal. Randomly distributed NV centers can be optically located on the xy plane, after which the SOMAmer set is placed at the same position.

FIG. 10 represents the steps performed in the exemplary method 1000 for detecting a target molecule in a sample solution and may not represent the complete process or all steps of this method. The various steps of Method 1000 are described below and are shown in FIG. 10, but these steps need not necessarily be all performed, and may be performed simultaneously or in a different order other than the order shown.

In step 1002, the method comprises contacting the sample solution with the capture reagent. The capture reagent is configured to adhere to a surface (eg, a crystalline film or substrate, or the surface of a single crystal diamond) and bind to the desired target molecule. Capturing reagents can be described as being captured by a surface, bound to a surface, attached to a surface, tethered to a surface, and / or anchored to a surface. The target molecule can be any target molecule having the property of binding to a capture reagent, including, but not limited to, the target molecule described in the present disclosure, such as a protein. Similarly, the scavenger reagent can be any suitable scavenger. This includes any of the capture reagents described in the present disclosure, such as, but not limited to, aptamers, nucleic acid molecules, or nucleic acid molecules having at least one 5-position modified pyrimidine (such as SOMAmer). .. In some examples, at least two different 5-position modified pyrimidines are attached to the surface, including at least one 5-position modified uridine and at least one 5-position modified cytidine.

In step 1004, the method comprises irradiating the excitation light with a color center located near the surface (eg, at a fixed depth in the crystal film). The excitation light is configured to induce fluorescence radiation (eg, through selection of the wavelength or bandwidth of the center) by the color center. In some examples, the color center is the nitrogen-vacancy center of the diamond crystal, and this surface is the surface of the diamond crystal.

In step 1006, the method measures the intensity of fluorescence emission using one or more detectors (eg, the integrated detection and treatment layer photodetector described above in connection with FIG. 9). including.

At step 1008, the method comprises detecting a change in the intensity of fluorescent radiation. The change in intensity depending on the binding of the target molecule to the capture reagent, and thus the change in the released intensity, indicates the presence of the target molecule.

Optionally, in step 1010, the method irradiates the color center with microwave radiation at a frequency that can induce the conversion of ground state electrons at the color center from the first substate to the second substate. May include. For example, microwave radiation may include a resonant frequency corresponding to the energy difference between the ground state zero-spin substate and the ground state non-zero-spin substate. Detecting changes in the intensity of fluorescent radiation in response to binding of target molecules in step 1008 identifies the resonance behavior of the color center based on the relationship between the measured intensity of fluorescent radiation and the frequency of microwave radiation. Can further include doing. For example, binding of the target molecule can induce a change in resonant frequency between the first and second lower states. Changes in resonance frequency can be identified based on the intensity of fluorescent radiation measured while varying the frequency of microwave radiation.

FIG. 11 represents the steps performed in the exemplary method 1100 for measuring the concentration of a target molecule and may not represent the complete process or all steps of this method. The various steps of Method 1100 are described below and are shown in FIG. 11, but these steps need not necessarily be performed and may optionally be performed simultaneously or in a different order than indicated.

In step 1102, the method exposes the fluid to the surface of a crystalline membrane (eg, especially a diamond membrane) to bind the capture reagents attached to the surface to a target molecule (eg, especially a protein) in the fluid. include. The crystal film contains at least one color center, such as a nitrogen-vacancy center or other suitable defect.

At step 1104, the method comprises irradiating the crystal membrane with excitation light configured to induce fluorescence radiation from at least one color center within the membrane. Optionally, step 1104 comprises irradiating the color center with microwave radiation having a frequency capable of inducing the conversion of ground state electrons at the color center from the first substate to the second substate. ..

At step 1106, the method comprises detecting changes in the nature of the color center based on fluorescence emission. The change in properties at the color center occurs in response to the binding of the target molecule by the capture reagent. In some examples, the capture reagent comprises a magnetic spin label such that the bond between the target molecule and the capture reagent changes the magnetic field at the color center. Changes in properties at the color center can be caused, at least in part, by changes in the magnetic field.

At step 1108, the method comprises determining the concentration of the target molecule in the fluid based on the change in properties at the detected color center. For example, the characteristic of the detected change can represent the number or approximation of the target molecules bound to the surface of the membrane by the capture reagent, and the concentration of the target molecules in the fluid can be derived from the number of target molecules and the volume of the fluid. Can be estimated.

FIG. 12 represents the steps performed in the exemplary method 1200 for detecting a target molecule and may not represent the complete process or all steps of this method. The various steps of method 1200 are described below and are shown in FIG. 12, but these steps do not necessarily have to be performed and may optionally be performed simultaneously or in a different order than indicated.

In step 1202, the method exposes the crystalline substrate (eg, a diamond membrane) to the sample solution so that the trapping reagent attached to the surface of the substrate can bind to the target molecule that is expected to be present in the sample solution. include. The crystalline substrate contains at least one color center, such as a nitrogen-vacancy center in diamond. The target molecule can be a protein or any other target molecule described in any of the present disclosures. Similarly, the capture reagent can be any molecule suitable for specifically binding to the desired target molecule and can include aptamers, SOMAmer, and the like. In some examples, the capture reagent is an aptamer containing at least two distinctly different 5-position modified pyrimidines.

At step 1204, the method comprises irradiating the crystalline substrate with electromagnetic radiation. Electromagnetic radiation is configured to stimulate or induce fluorescence by the color center within the crystal substrate. The characteristics of electromagnetic radiation, including center frequency, bandwidth, intensity, pulse energy, pulse duration, and / or polarization, can be designed to, at least in part, determine the characteristics of the emitted fluorescence.

At step 1206, the method comprises detecting fluorescence emitted by a color center, for example by one or more photodetectors.

At step 1208, the method comprises identifying changes in the properties of one or more color centers resulting from the binding of one or more target molecules from the sample solution to the capture reagent. For example, identifying this change can include identifying changes in the spectrum of radiation emitted by the color center, eg, detecting fluorescent radiation emitted by the color center, and the intensity of the emitted fluorescence, It may include identifying transient variations, or changes in the spectrum.

FIG. 13 represents the steps of exemplary method 1300 for making an apparatus for detecting the presence of protein molecules in a sample solution, which may not represent the complete process or all steps of this method. The various steps of method 1300 are described below and are shown in FIG. 13, but these steps need not necessarily be performed and may optionally be performed simultaneously or in a different order than indicated. Aspects of Method 1300 can be used, for example, to produce the integrated biochip shown in FIG.

At step 1302, the method comprises producing a crystal film, for example by chemical vapor deposition. For example, a ¹² C-rich diamond film can be produced by plasma CVD (plasma-enhanced chemical vapor deposition) of a mixture of methane and hydrogen gas on a diamond substrate.

At step 1304, the method comprises embedding a substituted atom in the crystal film. Substitution atoms are suitable for placement with vacancies in the crystal film to form a color center.

At 1306, pores are created in the crystal membrane, for example by colliding an ion beam and / or an electron beam on the membrane.

At step 1308, the method comprises creating a color center in the crystal film by arranging at least some pores and at least some substitution atoms together. Placing the substituted atoms and vacancies together can include, for example, baking the membrane at high temperature or by any other suitable process.

At step 1310, the method comprises attaching a capture reagent to a first surface on the crystal film. Step 1310 may include attaching the capture reagent to a portion of the surface region of the crystal membrane and passivating the region of the surface that is not bound to the capture reagent against non-specific binding by unwanted proteins. .. Passivation of other regions of the surface by attaching a capture reagent is a passivation molecule on the surface using some of the passivation molecules that contain active groups for binding to a particular target molecule. It can be achieved by binding, for example, a hydrophilic polymer). For example, some of the passivated molecules have a first end configured to bind to the surface and a second end having an active group configured to bind to DNA or another target molecule. Can have a part.

At step 1312, the method comprises attaching a photodetector to a second surface of the crystal film so that the photodetector can detect emission from the color center. The photodetector may be made using lithography and may be adhered to the crystal film using an optically transparent and mechanically rigorous adhesive. In some examples, the filter layer (eg, filter layer 904) is included between the photodetector and the membrane.

A typical detection scheme for measuring the concentration of a target molecule in a sample solution is to measure the intensity of fluorescence emission by at least one color center, fluorescence emission depending on the binding of at least one target molecule to a capture reagent. Includes identifying changes in the target molecule and determining the concentration of the target molecule in the sample solution based on the identified changes. For example, excitation and emission aspect of the nitrogen vacancy center represented in FIG. 7, the relaxation time T ₁ is can be included in the exemplary method is monitored completely by an optical method. T ₁ is measured by irradiating the NV center with excitation light to polarize the spin state of the basal to a lower level of _{m s = 0.} As shown in FIG. 4, _{the fraction of electrons excited from the lower level of m s} = ± 1 returns to the ground state via the dark transition, which causes the transition to the lower level _{of m s = 0.} Provides maximum strength. T ₁ is determined by periodically monitoring the fluorescence intensity after the excitation-mediated polarization is interrupted to see how quickly the ground state returns to a completely unpolarized state. An exemplary method for measuring protein in urine comprises the following steps, which can be performed simultaneously or in a different order other than the order shown.
1. 1. Prepare multiple calibration samples of urine containing known concentrations of the protein of interest.
2. These calibrated samples are flushed over the detection surface of the diamond layer to provide the protein with ample opportunity to bind to the attached SOMAmer and optionally recirculate the sample solution.
3. 3. The cleaning solution is flushed over the fluid contact surface of the diamond layer, providing the cleaning solution with ample opportunity to remove molecules that bind to SOMAmer in a non-specific manner, and optionally recirculate the cleaning solution.
4. The NV center is irradiated with a series of pulses of excitation light, each pulse having a duration long enough to result in complete polarization of the _{NV center to the ms = 0 state.} Time interval tau _i between these excitation pulses varies.
5. Fluorescence intensity immediately after start of the excitation pulse, and spin state is measured reference intensities employed as a target terminal of the excitation pulse at the time of being completely polarized to the lower level of the m _{s =} 0.
6. The normalized intensity is calculated by dividing the fluorescence intensity at invocation of each excitation pulse by the corresponding reference intensity.
7. The response plots of each calibration sample, by plotting the reference intensity of each pulse p _i for interval time t _i immediately preceding pulse p _i, to produce.
8. _{The relaxation time T 1} of each calibration sample is found by matching the decaying exponential function of form normalized intensity = I ₀ * exp (−τ / T _1).
9. To create a calibration curve of protein concentration vsT _1.
10. To generate a response plot, steps (2)-(8) are repeated using the sample of interest instead of the calibration sample.
11. The calibration curve of step 9 is applied to determine the protein concentration of each sample of interest.

Further methods may include, for example, the system shown in FIG. Optical detection of spin states is preferred, but the addition of microwave excitation and the introduction of static fields expands all conventional EPR-derived techniques for manipulating spin states. These examples (eg Hahn Echo, DEER, etc.) have been described above. There are numerous variations on the method described above, with the addition of additional spin manipulation steps via techniques well known in the art that may be selected to optimize the detection of bound analytes.

Although the given example focuses primarily on urine or blood (serum or crystals) as the sample solution, the biochips of the art work with various biological fluids or solutions derived from them, for example by dilution. Can be done. These are, but are not limited to, blood, plasma, serum, urine, semen, saliva, cerebrospinal fluid, sheep membrane fluid, glandular fluid, lymph fluid, papillary aspirate, bronchial aspirate, synovial fluid, joint aspirate, leukocyte. , Peripheral blood mononuclear cells, sputum, breath, cells, cell extracts, feces, tissues, tissue extracts, tissue biopsies, and cerebrospinal fluid.

Illustrative Combinations and Additional Examples This section describes further aspects and mechanisms of the proteome assay, presented without limitation as a series of paragraphs, some of which are benign and efficient. Can be represented alphabetically for sex. Each of these paragraphs may be combined in any suitable manner with the disclosure in any of the present applications comprising one or more other paragraphs and / or any substance incorporated by reference. Some of the following paragraphs clearly refer to other paragraphs and further limit and provide some, but not limited to, examples of suitable combinations.

A. A device for the detection of one or more species of target molecules in a fluid.
a) A solid support with a surface and
b) A capture reagent of one or more species that adheres to the surface.
c) Capture reagents and capture reagents that selectively bind to target molecules of a particular species.
d) With a color center located near the surface of the solid support,
e) An apparatus comprising a detector for detecting changes in the properties of the color center upon binding of a protein molecule to the capture reagent.

A1. The apparatus according to paragraph A, wherein the fluid comprises a biological fluid and the target molecule is a protein.

A2. The apparatus according to paragraph A1, wherein the capture reagent is an aptamer.

A3. The apparatus according to paragraph A2, wherein the aptamer is a nucleic acid molecule.

A4. The apparatus according to paragraph A3, wherein at least a portion of the nucleic acid molecule has at least one 5-position modified pyrimidine.

A5. The apparatus according to paragraph A4, wherein the solid support is a diamond crystal and the color center is a nitrogen-vacancy center.

A6. The apparatus according to paragraph A5, wherein the detector is an optical system that irradiates the nitrogen-vacancy center with radiation of a first range of wavelengths to detect wavelengths of a second range.

A7. The device according to paragraph A6, wherein the light source is a pulsed light source.

A8. The apparatus according to paragraph A7, wherein the change in the nature of the nitrogen-vacancy center is a change in the nature of the magnetic resonance of the nitrogen-vacancy center.

A9. The device of paragraph A8, further comprising a magnetic field source for providing a magnetic field.

A10. A device according to paragraph A9, further comprising a microwave source for providing microwave radiation.

A11. The device according to paragraph A10, wherein the microwave source is tuned to a frequency that resonates with the lower level of the ground state of the electrons at the nitrogen-vacancy center.

A12. The device according to paragraph A11, wherein the microwave source is a pulsed source.

A13. The apparatus according to paragraph A5, wherein the surface of the solid support is the {111} surface of a single crystal diamond.

A14. The apparatus according to paragraph A5, wherein the surface of the solid support is the {100} surface of a single crystal diamond.

The device according to paragraph A5, wherein the nitrogen-vacancy center is located within the surface of a 25 nanometer diamond crystal.

A16. The apparatus according to paragraph A7, wherein the nucleic acid molecule further comprises a spin label.

A17. The apparatus according to paragraph A12, wherein the nucleic acid molecule further comprises a spin label.

B. A device for simultaneously quantifying a single or multiple analytes in a sample solution.
a) A thin crystalline layer containing a color center defect 5 to 25 nanometers away from the surface where one surface is in contact with the sample liquid and in contact with the fluid.
b) Described in (a) such that the binding of the analyte to the binder affects a local magnetic field external to the color center defect, thereby altering the behavior of the color center defect in a detectable manner. A scavenger that adheres to the surface of the crystal layer that comes into contact with the fluid in the immediate vicinity of the color center defect.
c) An apparatus comprising a means for detecting a change in the behavior of the color center defect.

B1. The change in the behavior of the color center defect includes the change in fluorescence, and the means for detecting the change in fluorescence is
a) An optical filter layer coupled to the non-contact surface of the crystal layer to allow fluorescence radiation from color center defects to pass while excluding excitation light or other light.
b) A detection layer that is attached to an optical filter layer on the opposite side of the crystal layer for capture and quantification of fluorescence emitted by a color center defect, including intensity.
c) The apparatus according to paragraph B, comprising means for introducing excitation light into the crystal layer such that the color center defect is stimulated to emit fluorescence.

B2. The means for introducing the excitation light into the crystal layer is to introduce the light through the edge of the crystal layer so that the excitation light moves in a direction parallel to the surface of the crystal layer in contact with the fluid. The device according to paragraph B1.

B3. The change in the behavior of the color center defect includes the change in fluorescence, and the means for detecting the change in fluorescence is
a) An optical waveguide containing an optional optical filter coupled to the non-contact surface of the crystal layer to allow fluorescence radiation from color center defects to pass while excluding excitation light or other light.
b) A detection layer located opposite the optical waveguide from the crystal layer for capture and quantification of fluorescence emitted by color center defects, including intensity.
c) The apparatus according to paragraph B, comprising means for introducing excitation light into the crystal layer such that the color center defect is stimulated to emit fluorescence.

B4. Changes in the behavior of the color center defect include changes in an electric or magnetic field local to the color center defect, and means for detecting the change in the electric or magnetic field includes an electronic or photoelectron detector. The device according to paragraph B.

B5. The apparatus of paragraph B1, further comprising means for introducing microwave radiation into the crystal layer to influence and / or monitor the resonant behavior of the color center defect.

B6. The apparatus according to paragraph B5, comprising means for imposing a constant or various magnetic fields over a region containing a color center defect to correct resonance behavior.

B7. The apparatus according to paragraph B5, wherein the thin crystal layer is a diamond film and the color center defect is a nitrogen-vacancy center.

B8. The device according to paragraph B7, wherein the device can be regenerated for multiple uses.

B9. A binder specific for the analyte is bound to a magnetic spin label to increase its action upon binding of the analyte on a local magnetic field external to the color center defect, thereby binding the color. The device according to paragraph B8, which modifies the behavior of a central defect so that it can be detected.

B10. The apparatus according to paragraph B9, wherein the binder specific for the analyte is an aptamer or SOMAmer.

B11. The apparatus according to paragraph B9, wherein the optical filter layer is composed of a layered dielectric film to form a dichroic bandpass filter.

B12. The detection layer is composed of a CMOS avalanche photodetector that makes immediate contact with the optical filter layer, and a signal from the photodetector passes through a high-speed electron gate and a signal passing through the high-speed gate. The device of paragraph B9, wherein the data passed through the event counter and obtained as a result collected by the event counter is delivered to the processor for analysis.

B13. The set of tethered SOMAmers is identical in their specificity to a specific analyte and is measured with the set of nitrogen-vacancy centers that collectively emit fluorescence captured by individual photodetectors. The device according to paragraph B9, which interacts in a possible way.

B14. The apparatus according to paragraph B13, wherein the set of SOMAmers consists of a single molecule and the set of nitrogen-vacancy centers consists of a single center.

B15. The apparatus according to paragraph B13, wherein the SOMAmer set comprises a plurality of molecules that are identically specific to the same analyte, and the nitrogen-vacancy center set consists of a single center.

B16. The apparatus according to paragraph B13, wherein there are multiple sets of tethered SOMAmers, each of which is specific to a different analyte.

B17. The apparatus according to paragraph B16, wherein the number of tethered SOMAmer sets is 100-10,000.

B18. Means for orienting the fluid in contact with the fluid in the diamond layer, including means for recirculating the fluid as needed, are included.
a) Sample solution and
b) A cleaning solution used to remove molecules that bind in a non-specific manner to the surface of the SOMAmer or diamond layer in contact with the fluid.
c) Bonds in both specific and non-specific forms to the SOMAmer or the surface of the diamond layer in contact with the fluid without causing damage or modification to the surface of the SOMAmer or the diamond layer in contact with the fluid. The apparatus according to paragraph B13, comprising a regenerating fluid used to remove the molecules that form.

B19. The apparatus according to paragraph B18, wherein the sample solution is a biological fluid derived from a human subject.

B20. The device according to paragraph B19, wherein the biological fluid is urine.

B21. The device according to paragraph B20, wherein the device is contained within a toilet, toilet bowl, or urine container, thereby being designed to allow capture and analysis of urine from the toilet, toilet bowl, or urine container. Device.

B22. The device according to paragraph B21, which compares the data produced by the device with a database of proteomics for diagnosing possible disease states.

B23. The method by which the concentration of the analyte is described:
a) To prepare multiple calibration samples of the fluid containing the known concentrations of the above article.
b) The analysis provides sufficient opportunities to orient the calibration sample to the fluid-contacting surface of the diamond layer, including optionally recirculating the calibration sample solution, and to bind to the tethered SOMAmer. To provide things and
c) Directing the cleaning solution to the surface of the diamond layer in contact with the fluid, giving the cleaning solution sufficient opportunity to remove molecules that bind to the SOMAmer in a non-specific manner. Opportunity is optional, including orienting, including recirculation of the cleaning fluid,
d) Irradiating the nitrogen-vacancy center with excitation light, including the frequency at which the light induces fluorescence emission at the nitrogen-vacancy center, and measuring the fluorescence emission intensity. To do and
e) Irradiating the nitrogen-vacancy center with microwave radiation, where the frequency of the microwave radiation changes from a spin state of 0 to a spin state of ± 1 at the nitrogen-vacancy center. Irradiation and irradiation, which fluctuate over a range predicted to include a resonance frequency that induces the conversion of ground-state electrons.
f) For each of the above calibration samples, make a plot of the fluorescence emission intensity vs. the microwave emission frequency.
g) To create a calibration curve for some selected features of the plot of the above calibration sample from the analyte concentration pair (f).
h) Repeating steps (b)-(f), using the sample of interest instead of the calibration sample, to create the plot as in (f).
i) The apparatus of paragraph B18, wherein the calibration curve of step (f) is applied and measured in the sample solution according to determining the concentration of each analyte of the sample of interest.

B24. The apparatus and method according to paragraph B13, wherein the selected feature of the plot is the resonant microwave frequency, which is determined by positioning the most extreme local minima.

B25. The apparatus and method according to paragraph 23, wherein the selected features of the plot represent the width of the resonant inversion peak in some form, such as full width at half maximum (FWHM), or coefficient of variation (CV).

B26. The method by which the concentration of the analyte is described:
a) To prepare multiple calibration samples of the fluid containing the known concentrations of the above article.
b) The analysis provides sufficient opportunities to orient the calibration sample to the fluid-contacting surface of the diamond layer, including optionally recirculating the calibration sample solution, and to bind to the tethered SOMAmer. To give things and
c) Directing the cleaning solution to the surface of the diamond layer in contact with the fluid, giving the cleaning solution sufficient opportunity to remove molecules that bind to the SOMAmer in a non-specific manner. Opportunities include, optionally, recirculation of the above cleaning fluids, and
d) a pulse p _i of the rectangular wave of a series of excitation light the nitrogen - the method comprising irradiating a vacancy center, the light is, the nitrogen - containing a frequency that induces fluorescence emission vacancy centers and each pulse and long enough to provide a complete polarization of the 0 spin state of vacancy center, irradiating, to further vary the spatial tau _i between these the Symbol excitation pulse p _i, - but each the nitrogen
e) The fluorescence intensity immediately after the activation of each of the excitation pulses and the reference intensity taken at the end of each excitation pulse are measured, the reference intensity being completely in the zero spin state. To measure, to be measured when it should be polarized,
calculating a normalized intensity by f) at said corresponding reference intensity dividing the fluorescence intensity at start of each excitation pulse p _i,
By plotting the reference intensity of each pulse p _i for g) pulse p _i interval time immediately preceding the t _i, and making a response plot for each calibration sample,
h) formula _{y = I 0 * exp (-τ} / T1) ( wherein y is dependent variables consisting of a set of normalized intensity of each pulse _{p i,} the independent variables tau, the pulse by fitting an exponential function to decay of p is the set of intervals preceding time each _i), and finding the calibration samples each relaxation time T _1,
i) a creating a calibration curve of known analyte concentration versus step (h) from the relaxation time T ₁ of the in the calibration sample,
j) Repeat steps (b)-(h) using the sample of interest instead of the calibration sample to create a plot as in (g), and relax as in step (h). Finding time and
k) The apparatus of paragraph B18, wherein the calibration curve of step (i) is applied and measured in the sample solution according to determining the concentration of the analyte for each sample of interest.

C. A device for detecting target molecules
With a surface calibrated to contact the fluid,
A plurality of capture reagents attached to the surface, each configured to bind to a target molecule,
With multiple color centers located proximal to the surface,
An apparatus comprising at least one detector configured to detect a change in at least one property of a color center in response to binding of a target molecule to one of the capture reagents.

C1. The apparatus according to paragraph C, wherein the target molecule is a protein and the capture reagent is an aptamer.

C2. The device according to paragraph C1, wherein the aptamer is a nucleic acid molecule.

C3. The apparatus according to paragraph C2, wherein the nucleic acid molecule has at least one 5-position modified pyrimidine.

C4. The apparatus according to paragraph C, wherein the surface is the surface of the diamond crystal and the color center is the nitrogen-vacancy center of the diamond crystal.

C5. The apparatus according to paragraph C4, wherein the diamond crystal is a single crystal diamond.

C6. Further including a light source configured to irradiate the nitrogen-vacancy center with radiation having a wavelength in the first range, the detector is configured to detect radiation having a wavelength in the second range. , The apparatus according to paragraph C4.

C7. The apparatus according to paragraph C4, wherein the above properties are related to magnetic resonance at the nitrogen-vacancy center.

C8. The apparatus of paragraph C4, further comprising a microwave source configured to provide microwave radiation having a frequency in the range including the lower level resonant frequency of the ground state of the nitrogen-vacancy center electron.

C9. The apparatus according to paragraph C, wherein the capture reagent comprises reagents belonging to a plurality of capture species, and each capture species is configured to bind to a target molecule of a particular target species.

C10. A device for measuring the concentration of target molecules
A crystal film containing at least one color center and
A plurality of capture reagents that are attached to the surface of the crystal film and are configured to bind to the target molecule.
A device comprising a detector assembly configured to illuminate the color center with excitation light and detect the emission of electromagnetic radiation from the color center.

C11. The apparatus according to paragraph C10, wherein the capture reagent comprises a magnetic spin label and the magnetic field at the center of color changes in response to binding of a target molecule to one of the capture reagents.

C12. The detector assembly is configured to illuminate the color center with microwave radiation having a frequency capable of inducing the conversion of ground state electrons at the color center from the first substate to the second substate. The device according to paragraph C11.

C13. The apparatus according to paragraph C10, wherein the crystal film is a diamond film and the color center is a nitrogen-vacancy center.

C14. Detection of electromagnetic radiation from the color center by binding of one of the capture reagents to one of the target molecules by altering the interaction between the spin label of the capture reagent and the color center. The device according to paragraph C10, which brings about possible changes.

C15. A device for detecting target molecules
Multiple capture reagents that adhere to the surface of the crystal substrate and are configured to capture the target molecule from the sample solution.
A plurality of color centers located at a fixed distance from the capture reagent, wherein the fixed distance is such that at least one property of the color center depends on the capture of one of the target molecules by one of the capture reagents. With multiple color centers, small enough to change,
A device comprising a detector configured to detect a change in at least one property of the color center.

C16. The apparatus according to paragraph C15, wherein the capture reagent is an aptamer.

C17. The apparatus according to paragraph C16, wherein the capture reagent is an oligonucleotide.

C18. The apparatus according to paragraph C16, wherein the capture reagent is a nucleic acid molecule having a pyrimidine modified at least at the 5-position.

C19. The apparatus according to paragraph C18, wherein the crystal substrate is a diamond film and the color center is a nitrogen-vacancy center.

D0. A method for detecting target molecules in a sample solution.
Contacting the sample solution with a capture reagent configured to adhere to the surface and bind to the target molecule.
Irradiating the color center located proximal to the surface with excitation light configured to induce fluorescence radiation by the color center.
Measuring the intensity of the fluorescent radiation using one or more detectors,
A method comprising detecting a change in the intensity of the fluorescent radiation in response to binding of the target molecule to the trapping reagent.

D1. Further including radiating the color center with microwave radiation at a frequency capable of inducing the conversion of ground state electrons at the color center from the first substate to the second substate.
Detecting changes in the intensity of the fluorescent radiation in response to the binding of the target molecule to the trapping reagent is based on the relationship between the measured intensity of the fluorescent radiation and the frequency of the microwave radiation. Including identifying the resonance behavior of
The method according to D0.

D2. The method according to paragraph D0 or D1, wherein the target molecule is a protein.

D3. The method according to any one of paragraphs D0 to D2, wherein the capture reagent is an aptamer.

D4. The method according to any one of paragraphs D0 to D3, wherein the capture reagent is a nucleic acid molecule having a pyrimidine modified at least at the 5-position.

D5. The method according to any one of paragraphs D0 to D4, wherein the surface is the surface of the diamond crystal and the color center is the nitrogen-vacancy center of the diamond crystal.

D6. The capture reagent is an aptamer comprising at least one first five-position modified pyrimidine and at least one second five-position modified pyrimidine, and the first five-position modified pyrimidine and The second 5-position modified pyrimidine is a different 5-position modified pyrimidine.
The first 5-position modified pyrimidine is a 5-position modified uridine, and the second 5-position modified pyrimidine is a 5-position modified cytidine, or the above-mentioned first The 5-position modified pyrimidine of 1 is the 5-position modified cytidine, and the second 5-position modified pyrimidine is the 5-position modified uridine.
The method according to any one of paragraphs D0 to D5.

E0. A method for measuring the concentration of a target molecule,
By exposing the fluid to the surface of the crystal film and binding the capture reagent attached to the surface to the target molecule in the fluid,
Irradiating the film with excitation light configured to induce fluorescence radiation by at least one color center in the crystal film.
Based on the fluorescence radiation, detection of changes in the properties of the color center depending on the binding between the target molecule and the capture reagent, and
A method comprising determining the concentration of a target molecule in the fluid based on the detected change.

E1. The method according to paragraph E0, wherein the target molecule is a protein.

E2. The method of paragraph E1 or E2, wherein the capture reagent comprises a magnetic spin label and the magnetic field at at least one color center changes depending on the binding between the target molecule and the capture reagent.

E3. Irradiating the film with the excitation light irradiates the color center with microwave radiation having a frequency that can induce the conversion of electrons in the ground state at the color center from the first lower state to the second lower state. The method according to any one of paragraphs E0 to E2, comprising irradiating.

E4. The method according to any one of paragraphs E0 to E3, wherein the color center is the nitrogen vacancy center.

E5. The method according to any one of paragraphs E0 to E4, wherein the crystal film is a diamond film.

F0. A method for detecting target molecules
The crystal substrate is exposed to the sample solution so that a plurality of capture reagents attached to the surface of the crystal substrate bind to the target molecule in the sample solution.
A method comprising identifying changes in the properties of one or more color centers within the crystal substrate in response to binding of the target molecule to the capture reagent.

F1. The method according to paragraph F0, wherein the crystal substrate is a diamond film and the color center is a nitrogen-vacancy center in the diamond film.

F2. Paragraph F0 or F1 further comprises irradiating the crystalline substrate with electromagnetic radiation, and identifying changes in the properties of one or more color centers comprises detecting fluorescent radiation emitted by the color centers. The method described in.

F3. The method according to any one of paragraphs F0 to F2, wherein the capture reagent is an aptamer containing at least two distinctly different 5-position modified pyrimidines.

F4. The method according to any one of paragraphs F0 to F3, wherein the target molecule is a protein.

G0. A method for manufacturing a device for detecting the presence of protein molecules in a sample solution.
Making a crystal film and
Embedding a plurality of substituents in the crystal film and
Creating a plurality of pores in the crystal film and
By arranging at least some substitution atoms together with at least some pores, a color center is formed in the crystal film, and
Adhering a plurality of capture reagents to the first surface of the crystal film and
A method comprising attaching a layer of photodetector to the second surface of the crystal film.

G1. The method of paragraph G0, wherein the crystal film is made using chemical vapor deposition.

G2. The method of paragraph G0 or G1, wherein the pores are made using an electron beam.

G3. The method according to any one of paragraphs G0 to G2, wherein the substituted atom is arranged together with the pores by baking the crystal film at a high temperature.

G4. Adhering the photodetector layer can be done by using lithography to make the photodetector layer and by using an optically transparent adhesive to the second surface of the crystal film. The method according to any one of paragraphs G0 to G3, comprising bonding layers of photodetectors.

Claims (20)

A device for detecting label-free target analytes,
With surfaces configured to come into contact with fluids,
At least two capture reagents attached to the surface, with at least two capture reagents configured such that each capture reagent binds to a different label-free target analyte.
With a plurality of color centers located proximal to the surface,
Includes at least one detector configured to detect changes in at least one property of the color center in response to one binding of the different label-free target analyte to the corresponding one of the capture reagents. Device. The apparatus of claim 1, wherein the different label-free target analyte is a protein and the capture reagent is an aptamer. The device according to claim 2, wherein the aptamer is a nucleic acid molecule. The apparatus according to claim 3, wherein the nucleic acid molecule has at least one modified pyrimidine at the 5-position. The apparatus according to claim 1, wherein the surface is the surface of the diamond crystal, and the color center is the nitrogen-vacancy center of the diamond crystal. The apparatus according to claim 5, wherein the diamond crystal is a single crystal diamond. A light source configured to irradiate the nitrogen-vacancy center with radiation having a wavelength in the first range is further included, and the detector is configured to detect radiation having a wavelength in the second range. The device according to claim 5. The device of claim 5, wherein the property relates to magnetic resonance at the nitrogen-vacancy center. 5. The fifth aspect of the invention, further comprising a microwave source configured to provide microwave radiation having a frequency in the range including the ground state resonance frequency of the nitrogen-vacancy center electron. Device. A device for measuring the concentration of label-free target analytes.
A crystal film containing at least one color center and
A plurality of capture reagents attached to the surface of the crystal film and configured to bind to different label-free target analytes.
To detect electromagnetic radiation emitted from one of the color centers upon binding of the corresponding different label-free target analyte to one of and said capture reagent to irradiate the color centers by the excitation light A device that contains a detector assembly configured in. 10. The apparatus of claim 10, wherein the capture reagent comprises a magnetic spin label and the magnetic field at the color center changes depending on the binding of a label-free target analyte to one of the capture reagents. The detector assembly is configured to illuminate the color center with microwave radiation having a frequency capable of inducing the conversion of ground state electrons at the color center from the first substate to the second substate. The device according to claim 11. The apparatus according to claim 10, wherein the crystal film is a diamond film and the color center is a nitrogen-vacancy center. One binding of the label-free target analyte to one of the capture reagents in the emission of electromagnetic radiation from the color center by altering the interaction between the spin label of the capture reagent and the color center. The device of claim 10, which produces a detectable change. A device for detecting label-free target analytes,
A plurality of capture reagents attached to the surface of the crystalline substrate and each configured to capture different label-free target analytes from the sample solution.
A plurality of color centers which are arranged in a substantially fixed distance from said capture reagent, prior Symbol least one color center properties one of the label-free target analyte according to one of the capture reagent you change depending on the capture, a plurality of color centers,
A device comprising a detector configured to detect a change in at least one property of the color center. The device according to claim 15, wherein the capture reagent is an aptamer. The device according to claim 16, wherein the aptamer is a nucleic acid molecule. The apparatus according to claim 17, wherein the nucleic acid molecule is a nucleic acid molecule having at least one 5-position modified pyrimidine. The apparatus according to claim 15, wherein the crystal substrate is a diamond film and the color center is a nitrogen-vacancy center. The apparatus according to claim 15, wherein the capture reagent is attached to the first substrate of the crystal substrate, and the detector is attached to the second surface of the crystal substrate.

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